Bottom Line:
However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease.Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells.Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant.

Affiliation: Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia, Australia.

ABSTRACTArginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.

Mentions:
The V2R couples to Gs protein, thereby activating adenylate cyclase and increasing intracellular cAMP. Accordingly, we tested the ability of the three mutant receptors to increase cAMP levels using a homogenous time-resolved fluorescence (HTRF) assay (Figure 1). Treatment of transiently transfected HEK293FT cells with AVP caused a robust dose-dependent increase in cAMP (Figure 1A), with particularly high potency for the wild-type and V266A mutant receptors, the pEC50 values of which were not significantly different (Table 1). In contrast, AVP was 29- or 427-fold less potent in cells transfected with M311V or R181C HA-V2R respectively (Table 1). Next, we tested for the ability of Rluc8-tagged V2R to couple to Gs in the same cell line (Figure 1B), as it is important to check that the addition of a BRET tag does not compromise normal receptor function [34], [35]. Again, AVP caused a dose-dependent increase in cAMP, and for each receptor, potency was comparable to that obtained with the HA-tagged V2R (Table 1). Furthermore, the ligand-induced β-arrestin recruitment shown below provides additional validation of V2R/Rluc8 functionality. We also tested whether a different cellular background influenced the effect of the mutations on cAMP signaling. No significant difference in potency was observed between wild-type and V266A mutant receptors expressed in COS7 cells compared to the HEK293FT cell line. Furthermore, a significantly lower (60-fold) AVP potency was again observed with M311V and significantly lower still with R181C (3890-fold lower than wild-type). Indeed, expression of the R181C receptor in COS7 cells resulted in a significantly lower potency of AVP compared to its expression in HEK293FT cells (Table 1).

Mentions:
The V2R couples to Gs protein, thereby activating adenylate cyclase and increasing intracellular cAMP. Accordingly, we tested the ability of the three mutant receptors to increase cAMP levels using a homogenous time-resolved fluorescence (HTRF) assay (Figure 1). Treatment of transiently transfected HEK293FT cells with AVP caused a robust dose-dependent increase in cAMP (Figure 1A), with particularly high potency for the wild-type and V266A mutant receptors, the pEC50 values of which were not significantly different (Table 1). In contrast, AVP was 29- or 427-fold less potent in cells transfected with M311V or R181C HA-V2R respectively (Table 1). Next, we tested for the ability of Rluc8-tagged V2R to couple to Gs in the same cell line (Figure 1B), as it is important to check that the addition of a BRET tag does not compromise normal receptor function [34], [35]. Again, AVP caused a dose-dependent increase in cAMP, and for each receptor, potency was comparable to that obtained with the HA-tagged V2R (Table 1). Furthermore, the ligand-induced β-arrestin recruitment shown below provides additional validation of V2R/Rluc8 functionality. We also tested whether a different cellular background influenced the effect of the mutations on cAMP signaling. No significant difference in potency was observed between wild-type and V266A mutant receptors expressed in COS7 cells compared to the HEK293FT cell line. Furthermore, a significantly lower (60-fold) AVP potency was again observed with M311V and significantly lower still with R181C (3890-fold lower than wild-type). Indeed, expression of the R181C receptor in COS7 cells resulted in a significantly lower potency of AVP compared to its expression in HEK293FT cells (Table 1).

Bottom Line:
However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease.Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells.Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant.

Affiliation:
Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia, Australia.

ABSTRACTArginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.